Native lignin extraction from soft- and hardwood by green and benign sub/supercritical fluid extraction methodologies

Lignin constitutes an impressive resource of high-value low molecular weight compounds. However, robust methods for isolation of the extractable fraction from lignocellulose are yet to be established. In this study, supercritical fluid extraction (SFE) and CO2-expanded liquid extraction (CXLE) were employed to extract lignin from softwood and hardwood chips. Ethanol, acetone, and ethyl lactate were investigated as green organic co-solvents in the extractions. Additionally, the effects of temperature, CO2 percentage and the water content of the co-solvent were investigated using a design of experiment approach employing full factorial designs. Ethyl lactate and acetone provided the highest gravimetric yields. The water content in the extraction mixture had the main impact on the amount of extractable lignin monomers (LMs) and lignin oligomers (LOs) while the type of organic solvent was of minor importance. The most effective extraction was achieved by using a combination of liquid CO2/acetone/water (10/72/18, v/v/v) at 60 °C, 350 bar, 30 min and 2 mL min−1 flow rate. The optimized method provided detection of 13 LMs and 6 lignin dimers (LDs) from the hardwood chips. The results demonstrate the potential of supercritical fluids and green solvents in the field of mild and bening lignin extraction from wood.


Introduction
Lignocellulosic biomass has become of great interest in the past few years for its potential to provide sustainable fuels and valuable chemicals. 1-3 Lignin, a major component of lignocellulosic biomass, is a biopolymer based on p-coumaryl alcohol (H-unit), coniferyl alcohol (G-unit), and sinapyl alcohol (S-unit). 4,5 It is usually produced as a byproduct of the wood pulping process, and most of the obtained lignin is directly burnt to recover energy. 6 Lignin is the most abundant renewable source of aromatic compounds, but methods capable of producing value from this biopolymer are currently underdeveloped. The chemical structure of lignin is still not completely resolved and depends on species and geographic origin. 7,8 Due to its complexity, heterogeneity and reactivity, it has been difficult to isolate lignin in its native form without introducing chemical modication. 9,10 Hence, lignin extraction from biomass is a key process 11,12 to provide high-value compounds and its chemical characterization is of fundamental importance. 13,14 Currently, the main methods used for lignin isolation are based on either lignin desolvation or hydrolysis of the polysaccharides, leaving lignin as an insoluble residue. 15,16 In the Kra and organosolv processes, the separation of lignin from the polysaccharide fraction is performed using basic and acidic conditions, respectively. On the contrary, in the Klason process the polysaccharides are hydrolysed by sulphuric acid and the lignin is le as a solid residue. 17 However, these processes require high temperatures and pressures, as well as long reaction times and therefore producing unwanted side-reactions as well as consuming signicant amounts of energy. [18][19][20] Due to these drawbacks, alternative and greener extraction methods have been explored based on e.g. microwaves 21,22 and ionic liquids. 23,24 Unfortunately, these methods oen involve the use of strong acids and/or high extraction temperature, which may alter the chemical structure of lignin.
Recently, supercritical uid technologies have been applied for the extraction of natural compounds from renewable resources such as plants, microalgae, seaweeds and also food by-products. [25][26][27][28][29] In the eld of lignin processing, subcritical and supercritical uids have been used in depolymerisation reactions 30,31 but so far not tested in methods for extraction of lignin from lignocellulosic biomass.
Supercritical uid extraction (SFE) is based on the use of a solvent at temperature and pressure above its critical point. Under this condition the uid gains a liquid-like density and a gas-like viscosity, which provide fast mass transfer, which oen is an advantage in separation processes. 32,33 CO 2 is the most commonly used solvent for SFE. The addition of a co-solvent, also known as organic modier, can be used to increase the relative permittivity of the uid, which is benecial for the extraction of polar compounds. 34 If the organic co-solvent is present in higher amounts in the mixture than the CO 2 (molar fraction >0.5), the uid can be dened as a CO 2 -expanded liquid (CXL). 35 CXLs have a tuneable relative permittivity and the presence of liquid CO 2 reduces the viscosity of the solvent, thereby enabling faster mass transfer, as compared to the pure co-solvent. [35][36][37] Thus, when the extraction mixture contains higher amounts of CO 2 the extraction is dened as SFE, while with lower amounts of CO 2 is a CXL extraction (CXLE).
Supercritical and subcritical CO 2 show attractive physicochemical properties to be used in extraction of lignin. This notwithstanding, SFE and CXL extraction (CXLE) methodology have not been tested for their ability to achieve mild and benign extraction of lignin monomers (LMs) and lignin oligomers (LOs) directly from wood.
In the present study, we investigate the applicability of SFE and CXLE, using different solvent mixtures based on CO 2 , ethanol, acetone, ethyl lactate, and water, for the extraction of lignin from two sources of woodchips.

Sample preparationcalibration solutions
Stock solutions of the LMs were prepared in acetone/water (70/ 30, v/v) at a concentration of 100 mg mL −1 and then diluted to produce calibration solutions in the range of 0.1-50 mg mL −1 . An internal standard stock solution containing o-vanillin, 3hydroxyacetophenone and 2-hydroxy-3-methoxybenzoic acid at 1 mg mL −1 each was used to spike both the LM stock solutions and extracted samples.

Sample preparationextraction
Woodchips were extracted using an analytical SFE system (Waters MV-10, Milford, MA, USA) consisting of a uid delivery module for pumping CO 2 and co-solvent, an oven for heating of the extraction vessels, an automated back pressure regulator, a make-up pump, and a fraction collector module, as illustrated in Fig. 1. All the extractions were carried out in dynamic (continuous-ow) mode. For each experiment, 0.501 ± 0.006 g of woodchips were weighted using an analytical balance (four decimals precision). The woodchips, having a particle size of approximately 1 mm, were sandwiched layered in the extraction vessel with glass beads. The heads of the CO 2 pump were cooled using a chiller operated at 4°C. The sample was mixed with glass beads and placed into a 10 mL stainless steel extraction vessel. The ow of the two pumps was controlled with the volumetric ratio between CO 2 and the cosolvent. The pressure and the make-up solvent ow rate were kept at 350 bar and at 0.2 mL min −1 , respectively. Temperature, CO 2 percentage and water content in the co-solvent were varied according to an experimental design (see below). Aer each extraction, the system was ushed for 5 min with a CO 2 /co-solvent mixture that was identical to the one used in the extraction. The collected extracts were evaporated to dryness under a gentle ow of nitrogen gas at room temperature to obtain dry samples that were weighed to determine the gravimetric yield. The solid residue was then re-dissolved in 1.5 mL of acetone/water (70/30, v/v) and then centrifuged at 14 000 rpm for 10 min. The supernatant was collected and stored at −80°C to minimise possible altering effects in the solvent mixture until analysis. An aliquot of the sample was spiked with the internal standard solution to a nal concentration of 100 mg mL −1 before UHPSFC/QTOF-MS analysis.

Hansen solubility parameters (HSP)
Hansen solubility parameters (d), describing the solubility by the similarity between the solute and the solvent, were calculated to provide a theoretical foundation for evaluating lignin solubility. Briey, similar HSP values of solvent and solute indicate high solubility. For each combination of solvents and lignin, HSP values for ambient conditions were obtained from the literature, 38,39 or, when unavailable, calculated using the soware HSPiP. 40 The temperature dependence was estimated by the Jayasri and Yaseen equation: 41 where T c is the critical temperature of the solvent, d T i is the HSP at the reference temperature (T i ) and d T f is the HSP at the investigated temperature (T f ).
For CO 2 , the effect of the pressure was calculated using the method of Williams et al.: 42 In these equations, the reference parameters are determined using the method of Huang et al. 43 to establish the combination of pressure and molar volume corresponding to the total solubility for pure CO 2 . The molar volume (V) at different pressure and temperature is then calculated by using the molar mass and the density of CO 2 at the desired pressure and temperature. For the mixture of solvents, each parameter was calculated as the weighted average of the pure solvent values according to the desired composition of the mixture. The relative energy difference (RED) between lignin and the various solvents was calculated by the following equation: where R a is the difference between the solubility parameters of two substances and R 0 is the maximum solubility parameter difference, which still allows the lignin to be dissolved in the solvent. RED values lower than 1 indicate high affinity. 38 Method optimization using design of experiment (DoE) A full factorial design (FFD) was applied to optimize the inuence of the temperature (40-80°C), the CO 2 percentage in the extraction mixture (10-90 vol%) and the water content in the cosolvent (0-20 vol%) on the extraction. The FFD was choosen instead of a for example a central composite design (CCD) or a box-Behnken design (BBD) to minimise the total number of required experiments. With three investigated factors and three center points the selected FFD has in total 11 experiments, while a BBD or a CCD with the same number of factors and center points, would have 15 or 17 experiments, respectively. Separate designs were performed for the co-solvents ethanol, acetone and ethyl lactate. The solvent proportions were chosen to ensure that the binary or ternary system generated was in a single liquid phase. 36,[44][45][46] However, literature data are lacking for the ternary mixture of CO 2 , ethyl lactate and H 2 O.
Partial least squares regression (PLS), using a linear interaction term, was used to evaluate the models with the gravimetric yield (wt%; dry weight of extract/initial sample weight × 100), the number of identied LMs and LDs, and the concentration of vanillin, vanillic acid, coniferyl aldehyde, ferulic acid, syringaldehyde, syringic acid, and sinapaldehyde as responses. The ow rate and the extraction time were kept at 2 mL min −1 and 15 min, respectively. In total 11 experiments (Table S1, ESI, †), including three centre point replicates, were performed for each design. Models were evaluated by investigation of the explained variance (R2) and the predictive ability (Q2), obtained by cross-validation. Models showing a Q2 < 0.4 and signs of overtting (R2 − Q2 > 0.35) were considered unreliable. Models were optimized by removing insignicant terms to yield the highest possible Q2 and lowest degree of overtting.
The ow rate and the extraction time were optimized. Triplicate experiments were performed at each ow rate investigated (1, 2 and 4 mL min −1 ). During the experiments a fraction of the extracts was collected every 10 min over a period of 50 min. The fractions were dried and the extraction kinetics were visualized by plotting the extracted amount at the ve time points (mg of dried extract) vs. time and solvent volume.

Analysis by UHPSFC/QTOF-MS
Quantitative and qualitative analysis of all extracts and standard solutions were performed using a modied method previously reported by our group using ultra-high performance supercritical uid chromatography coupled with quadrupoletime-of-ight mass spectrometry (UHPSFC/QTOF-MS). 47 Chromatographic separation was performed with a Waters ultra performance convergence chromatography (UPC 2 ) system (Waters, Milford, MA, USA) hyphenated via a ow splitter (AQUITY UPC 2 splitter, Waters) with a Waters XEVO-G2 QTOF-MS (Waters). The injection solvent was acetone/water (70/30 v/ v) and 4 mL of samples were injected. The separation was performed with a DIOL column (1.7 mm, 3 mm × 100 mm, Waters) equipped with a torus DIOL VanGuard pre-column (2.1 mm × 5 mm, 1.7 mm, Waters). The separation was achieved using a gradient elution with CO 2 (A) and methanol (B) as co-solvent with a ow rate of 2 mL min −1 . The elution gradient started with 0% B (vol%) and then ramped up to 8.5% B (vol%) until 2.5 min, then ramped up to 25% B (vol%) until 5.5 min, then held for 2 min and decreasing to starting composition in 0.5 min. The column temperature was 50°C and the backpressure was 130 bar. Methanol with 5 mmol per L ammonia was used as a makeup solvent, at a ow rate of 0.5 mL min −1 . Electrospray ionization (ESI) was performed in negative mode with a capillary voltage of 3.0 kV, a cone voltage of 20 V, a source temperature of 120°C, a desolvation gas temperature of 600°C, and a desolvation gas ow rate of 1200 L h −1 . The mass spectrometer was used in full scan and MS 2 mode. The m/z range was m/z 50-1200. For the MS 2 measurements a collisioninduced dissociation energy ramp from 20 to 35 V was used.

Evaluation of matrix effects
Possible matrix effects were examined for vanillin using the standard addition method. The sample was extracted in triplicate using the optimised method and then spiked with 0.1, 0.5, 1, 5, and 10 mg mL −1 of vanillin, followed by construction of a calibration curve. o-Vanillin in a concentration of 1 mg mL −1 was used as internal standard. The slope of the standard addition curve was compared with that of the external calibration curve by the Student's t-test. An F-test was used to ensure homoscedasticity of the data.

Classication of LMs and LOs
For the classication of LMs and LOs a non-targeted analysis strategy previously developed by our group was used. 48 The nontargeted analysis strategy is based on the combination of highresolution mass spectrometry with principal component analysis-quadratic discriminant analysis (PCA-QDA) classication models. First, a feature list including m/z values was created using the open-source soware MZmine 2. Information about the used settings to create the feature list are given in Table S2 (ESI †). Four different PCA-QDA classication models based on literature data for LMs, LDs, trimers (LTRs) and tetramers (LTEs), respectively, were used to determine the number of monomeric units in the features provided in the peak list. The classication models were based on the number of carbon atoms, hydrogen atoms, and oxygen atoms, as well as ve Kendrick mass defects with base units of typical functional groups found in lignin-related phenolic compounds. The functional groups were phenol (C 6 H 5 O), methoxy/primary alcohol (CH 3 O), carboxylic acid (CHO 2 ), aldehyde (CHO) and secondary alcohol (CH 2 O). If a m/z value was classied as a LM, LD, LTR or LTE, the ring double bound equivalent (RDB), the mass difference, the detected and theoretical 13 C/ 12 C-intensity ratios and MS 2 data were used for validation. The result from this classication method is the number of features found for each individual oligomer size (monomers, dimers, etc.).

Hansen solubility parameters
HSPs were employed to assess the suitability of several green solvents for lignin extraction. Table S3 (ESI †) reports HSP values for twelve green solvents. In order to obtain a wide range of dispersion, dipole-dipole, and hydrogen bonding interaction strengths, ve of these solvents were selected (CO 2 , water, ethanol, ethyl lactate, and acetone) for further investigation. The HSP values calculated for pure solvents and their mixtures were then compared with literature values for lignin. 49 Then, HSP values for solvents and solvent combinations used in the DoE were correlated with HSP values of some LMs (vanillin, syringaldehyde, vanillic acid, ferulic acid, coniferyl aldehyde, syringic acid and sinapaldehyde). HSP values of the DoE design space for acetone are given in Table 1 and those for ethanol and ethyl lactate in Table S4 (ESI †)

Optimisation of the extraction parametersgravimetric yields
The extraction methods were optimised independently for hardwood (oak) and sowood (r). For oak wood, the best gravimetric yields were obtained using ethyl lactate as a co-solvent and the highest yield in the design space was 17.5 weight%. This yield was 4% higher than that obtained for acetone and 7% higher than that for ethanol (p = 0.005). Unlikely, in the literature ethanol has been reported as one of the best co-solvents. For instance, Jiang et al. 50 extracted lignin from eucalyptus bers by using a ternary mixture of scCO 2 , ethanol and water obtaining a yield of 35.9%. However, they used a notably high temperature and extraction time, 180°C and 60 min, respectively, which could potentially degrade the lignin. The contour plots in Fig. 2, obtained from the oak extraction using each of the respective three co-solvents investigated, show CO 2 percentage vs. water content in the co-solvent at 60°C. Extraction temperature was, independent of the extraction solvent, without effect on the gravimetric yield for the oak sample ( Fig. 2 and 3). As shown in Fig. 2, the presence of water in the co-solvent increases the gravimetric yield in the case of acetone. In addition, lowering the CO 2 percentage (i.e., increasing the co-solvent amount and thereby also the overall water content of the solvent) has a positive effect on the gravimetric yield from oak wood. This is in line with the effects predicted from analysis of the HSP values (Tables 1 and S4 †), i.e. when the CO 2 content is only 10 vol%, the presence of water in the co-solvent yield d P and d H values that are closer to those reported for lignin. Indeed, when the CO 2 percentage is low, the relative permittivity of the mixture can be adjusted with water allowing the extraction of LMs and LOs. On the contrary, when the CO 2 is at 90%, the values of d P and d H remain almost the same with or without the presence of water in the co-solvent.
Furthermore, as shown in Fig. 3 there is an interaction effect between the CO 2 percentage and the water content for acetone. Presumably, this interaction results from hydrogen bonding (see d H in Table 1), which is quite strong with ethanol, followed by ethyl lactate and last acetone. In summary, for all three Fig. 2 Contour plots from the interaction model obtained using full factorial design showing the influence of the CO 2 percentage and water content in the co-solvent on the gravimetric yield of oak wood using ethanol and acetone as co-solvent. Ethyl lactate was excluded as provided a non-reliable model. Temperature was 60°C and pressure 350 bar. Gravimetric yield is expressed as dry weight percent. investigated co-solvents, the CO 2 percentage appears to be the most inuential factor impacting on gravimetric yield, which is likely governed by the water-elicited regulation of the relative permittivity.
The gravimetric yields obtained for sowood (r) were significantly lower than the yields obtained for hardwood (p = 4 × 10 −6 ). In this case, only ethyl lactate provided an acceptable model, with models for ethanol and acetone showing very low predictive ability (see Table S6, ESI †). The gravimetric yield was negatively impacted by CO 2 percentage and water content, while other factors and interactions were insignicant (see Fig. S1 and S2, ESI †).
In summary, the use of ethanol as a co-solvent gives the lowest gravimetric yields for both oak and r wood, while both acetone and ethyl lactate enable relatively high yields of 5-17.5 weight% from the wood. The CO 2 percentage should be as low as possible for both wood types, while the water content show wood typedependent effects. Acetone is the most promising solvent, since evaporation of ethyl lactate, due to its high boiling point (154°C), requires a time-consuming and energy-demanding evaporation aer the extraction, which may also increase the risk of thermal degradation of the target compounds. If evaporation is not needed, then for oak wood, the best extraction solvent in terms of gravimetric yield is CO 2 /ethyl lactate/water (10/72/18, v/v/v) at 60°C (and 350 bars).

Characterisation of lignin extracts
Gravimetric analysis provides a rough description of the extraction process, lacking information on compound selectivity and chemical composition of the extracts. Possibly, the presence of high amounts of water may lead to high gravimetric yields due to dissolution of a large proportion of the polysaccharides in the wood, while at the same time reducing the yield of lignin. For this reason, the extracts were analysed using a UHPSFC/ESI-QTOF-MS and a PCA-QDA method to classify, identify and quantify lignin-derived compounds.
All the samples turned out to be highly complex, containing more than hundred putative compounds each. Fig. 4 shows the loading plot and score plot for literature data 48 as well as for one of the oak centre points chosen as a representative sample. The PCA-QDA model provided lists of tentative LMs and LOs, which aer further validation were summed up and used as response in the DoE. A two-way ANOVA including the tested solvents and wood types as factors and the number of detected compounds as response was performed using the data obtained by the triplicate measurements form the DoEs center points. As shown in Table S8, † a signicant difference of the means is present for both solvents and wood type. Also in this case, the slopes obtained by plotting the predicted values vs. the observed values were compared to the slope of 1 showing no signicant difference among the two slopes (Table S10 †).
Our results show that oak wood extracts contained a signicantly higher number of compounds classied as LMs and LOs (p = 0.01), with the majority of LOs assigned as LDs, in comparison to r wood. On average, a similar number of compounds were obtained using acetone and ethyl lactate as cosolvent. Overall, models calculated for the number of identied LMs and LOs showed very low predictability. Only ethyl lactate provided reasonable predictability for both wood types, while acetone provided a reasonable model only for r.

Compound-specic extraction yield
To further characterise the extraction selectivity, a number of LMs were quantied and resulting DoE data modelled separately for each LM. For the quantication, the slopes of the external and internal calibration curves for vanillin did not differ as reported in Table S11 † (p > 0.05 at a 95% condence), indicating that there are no major matrix effects impacting on its quantication. Consequently, external calibration curves were used throughout this investigation. Linear regression parameters obtained for each calibration curve are summarised in Table S12 (ESI †). The calculated concentrations are reported in Table S13. † Overall, models showed poor predictability (Table S6, ESI †). Only syringaldehyde and sinapaldehyde provided models with an acceptable predictive ability. For these models, an increased water content in acetone and ethyl lactate was again found be benecial for increasing the yield of these LMs (Fig. 5 and S9 †). The percentage of CO 2 had a negative inuence on the yield of syringaldehyde and sinapaldehyde with acetone and with ethyl lactate as co-solvent. Again, we can compare the values of HSP of the solvent mixtures in the design space with those reported for LMs (Tables 1 and S4 †). The use of an extraction mixture with higher amount of water gives values for d P and d H similar to those of LMs, suggesting an enhanced extraction of these analytes in water-rich solvents. On average, ethyl lactate gives slightly higher extraction yields of the LMs than the other co-solvents tested. However, as previously noted, the high boiling point of ethyl lactate still support the use of acetone as a co-solvent.

Extraction kinetics
In order to nalize the optimisation of the method, effects of the extraction time and ow rate on the extraction were investigated using the selected solvent (CO 2 /acetone/water, 10/72/18, v/v/v, at 60°C and 350 bar (Fig. 6)). As the gravimetric yield was found to reect the yield of lignin in the preceding investigations, the former was selected as a response due to the convenience of collecting these data. The ow rate was varied to determine which ow rate that provides the fastest extraction rate with a minimum of sample dilution. Moreover, these experiments can be used to determine whether the extraction process is limited by solubility or by mass transfer.
As shown in Fig. 6, complete extraction was achieved within 30 min and the results indicate that the extraction process is controlled by mass transfer rather than solubility. The process is desorption/diffusion-controlled, which implies that running the extraction at higher ow rate will not increase the extracted amount per time unit. However, equilibration and pressurization of the system becomes exceedingly time-consuming when the ow rate is set to the lowest investigated level (1 mL min −1 ). For this reason, extraction at a ow rate of 2 mL min −1 for 30 min was chosen as the optimal condition.

Extraction of lignin from oak wood with the nal method
An oak wood sample was extracted in triplicate with the optimised method having a solvent composition of CO 2 /acetone/ water (10/72/18, v/v/v) at 60°C and 350 bars, for 30 min at 2 mL min −1 . The extracts were analysed by UHPSFC/ESI-QTOF-MS; the base peak ion chromatogram of one oak sample is shown in Fig. S10. † A total of 104 putative compounds were detected in the resulting data. These compounds were subsequently analysed with the PCA-QDA classication model for LMs and LOs (Fig. 7).
In total, 27 compounds were classied as LMs, 7 as LDs, 9 as LTRs and 7 as LTEs. Aer validation, 19 compounds were le, out of which 13 were LMs and 6 LDs. The identied LMs and LDs are listed in Table 2. As shown in the table, most of the detected LMs were also identied. However, for three LMs and for all LDs chemical structure could be assigned, which is mainly due to the lack of chemical standards. The concentration of LMs being present at concentrations above the method limit of quantication are presented in Table 2. Among the investigated compounds, vanillin and syringaldehyde are the most easily extractable compounds from oak wood.

Conclusions
In the presented work, a new SFE-and CXLE-based method for extraction of lignin from wood chips has been thoroughly developed and optimized. The method allowed the extraction of LMs and LDs from hardwood and sowood chips. Either acetone and ethyl lactate are good co-solvents within the investigated parameter ranges. Furthermore, the addition of water to the co-solvent is benecial for the extraction of lignin, while the extraction temperature showed no positive signicant inuence.
Future perspective of the work will be the extraction of lignin from various lignocellulosic matrices such as grass and different species of hardwood and sowood. In addition, the use of this method to study biomass subjected to pre-treatments would provide interesting information on the compositional modication of the extracted lignin. Moreover, a deeper investigation of the selectivity of the tested co-solvents would potentially allow us to achieve more accurate quantitative results both for gravimetric yield and LM concentration.

Author contributions
FN performed experimental work and wrote the rst dra of the manuscript. JP contributed to the experimental work and draing of the manuscript. ER, MS, PS and CT critically discussed results of the study. All authors contributed to writing the nal version of the manuscript.

Conflicts of interest
There are no conicts to declare.